The present invention relates to liposomal formulations of antisense oligonucleotides, methods of making such formulations, and methods of using them to treat cancer.
Chronic myeloid leukemia (CML) is an acquired clonal disorder involving the hematopoietic stem cell characterized by a prominent expansion of granulocytes. 90-95% of CML patients have a Philadelphia chromosome (Ph+) in the dividing bone marrow cells. The Ph+ chromosome results from a reciprocal translocation, t(9;22) (q34;q11), which relocates the c-abl protooncogene on chromosome 9 to the breakpoint cluster region (bcr) of chromosome 22. The bcr-abl hybrid gene encodes a novel p210.sup.bcr-abl fusion protein with tyrosine kinase activity. p210.sup.bcr-abl is of either L-6 (bcr exon II and c-abl exon "2" linkage or b2/a2 linkage) or K-28 linkage (bcr exon II and c-abl exon "2" linkage or b3/a2 linkage). p210.sup.bcr-abl is believed to be involved in the pathogenesis of the disease by promoting selectively the expansion of mature myeloid progenitor cells.
The disease divides into two clinical phases: an initial chronic phase, followed by a fatal blast crisis phase. The treatment of CML is very problematic. The established methods of treatment of CML are (1) interferon and (2) syngeneic or allogeneic bone marrow transplant. Only 25% of patients develop long-term remissions. Goldman and Calabretta found that antisense oligonucleotides directed to the translation initiation site of the bcr-abl mRNA induced a reduction of p210.sup.bcr-abl expression and suppressed the growth of Ph+ cells but not Ph- cells. Thus the use of antisense oligonucleotides may offer a new therapeutic approach to CML.
The two main obstacles in using antisense oligonucleotides to inhibit gene expression are: (a) cellular instability and (b) cellular uptake. Natural phosphodiesters are not resistant to nuclease hydrolysis; thus high concentrations of antisense oligonucleotides are needed before any inhibition effect is observed. Modified phosphodiester analogs, such as phosphorothioates and methyl phosphonates, have been made to overcome this nuclease hydrolysis problem, but they have not provided a completely satisfactory solution to the problem.
The cellular uptake of antisense oligonucleotides is low. To solve this problem, two different approaches have been used. One approach is to use high concentrations of antisense oligonucleotides. Even though this approach can increase the uptake of antisense oligonucleotides, it may also induce non-specific, toxic side effects. The other approach is to use physical techniques such as calcium-phosphate precipitation, DEAE-dextran mediation, or electroporation to increase the cellular uptake of oligos. These techniques are difficult to reproduce and are inapplicable in vivo.
There is a need for improved antisense compositions for use in treatment of disease, and also a need for processes for making such improved compositions.